The present invention relates generally to a method of preparing an immunogen comprising outer membranes from Campylobacter jejuni (hereafter designated Cj) and Campylobacter coli (hereafter designated Cc), inoculating animals with the immunogen, and detecting the desired hybridoma-producing antibodies; and to a composition comprising the immunogen. The invention is drawn further to hybridoma cell lines developed by this method to produce (1) monoclonal antibodies specific to Cj only and (2) monoclonal antibodies that recognize both Cj and Cc exclusively, and uses thereof.
Campylobacters cause more human gastroenteritis than any other food-borne pathogen. Campylobacter enteritis is caused by two closely related species, Cj and Cc, but Cj is responsible for greater than 98% of human disease and produces more severe symptoms. Cj, has been further subdivided into Cj subspecies jejuni (Cjj) and Cj subspecies doylei (Cjd). (In the remainder of this application, the term Cjj will be used to designate C. jejuni subspecies jejuni and Cjd to designate C. jejuni subspecies doylei. The term Cj (Campylobacter jejuni) will be used to describe both C. jejuni subspecies jejuni and C. jejuni subspecies doylei collectively.) Cjj and Cjd are closely related; however, Cjj is the predominant subspecies causing human illness and isolated from food sources. Cj and Cc cause acute diarrhea in humans with associated enteritis, particularly in developing countries. These infections are prevalent in infants under 1 year in age and rank as the third most common cause of acute diarrhea after Rotavirus and enterotoxigenic Escherichia coli. The various clinical patterns of disease suggest that Campylobacter spp. and, more specifically, Cj strains are diverse and may possess more than one type of virulence factor. In addition, Cj, unlike Cc, are more often associated with symptomatic infections and with bloody diarrhea.
Since Campylobacter enteritis is a considerable world health problem contributing to morbidity in developed countries and to high mortality rates in children in developing countries, it is of clinical importance to develop a specific and rapid diagnostic assay to identify pathogenic Campylobacter in the stool of enteritis patients. The available culture methods for detecting the organism generally are extremely time consuming and costly. Several studies have reported production of anti-Campylobacter MAbs and their use in immunoassays to detect Campylobacter infection (Chaiyaroj et al., 1995).
Campylobacter was not recognized as a human pathogen until the mid 1970""s. Previously, the principal food-borne pathogen of concern had been Salmonella spp. Since the 1970s, the food industry and the regulatory agencies have become aware of the importance of other food-borne pathogens, such as Yersinia enterocolitica, Escherichia coli O157:H7, and Listeria monocytogenes. Very recently, Cj has become recognized as the most frequent cause of gastroenteritis in the U.S. and has been observed as the most common pathogen associated with Guillain-Barre syndrome in humans. It is estimated that about 1 in 1,000 Campylobacter infections results in this serious illness (Nachamkin et al., 1992).
Campylobacter is very prevalent in poultry (Madden et al., 1998) and contaminates milk (Docherty et al., 1996). Hazard analysis critical control point (HACCP) systems for poultry are being implemented internationally (Notermans et al., 1994), and beginning in 1997 in the U.S., large poultry processors have been required to meet performance standards for the frequency and amount of Salmonella in their product (Anonymous, 1996). It is anticipated that similar performance standards will be established for Campylobacter spp. as better culture and quantitative detection methods are developed. The development of rapid, reliable, and cost-effective methods for the detection of food-borne pathogens is crucial to accurately assess the safety of a food product, the effectiveness of new control measures to minimize pathogens in a production or processing environment, and the basic biology and ecology of pathogens in the food environment.
Campylobacters are known to be widespread in the environment and contaminate soil and water (Stanley et al., 1998). Environmental monitoring requires detection methods that are field-based, portable, rapid, and capable of analyzing multiple samples. Biosensors that contain specific immobilized antibodies to capture a pathogen for subsequent detection by a laser light or other sensitive detection system are being developed (Zhou et al., 1998). The specificity, sensitivity, and affinity of the antibody in a biosensor are key factors for success. The following U.S. patents are incorporated by reference.
U.S. Pat. No. 4,404,194 discloses a 90 kDa protein from C. jejuni that has immuno-suppressive activity.
U.S. Pat. No. 4,785,086 discloses a DNA probe for detecting C. jejuni. 
U.S. Pat. No. 4,882,271, discloses a 300-700 kDa antigen from Campylobacter pylori and its use in various assays.
U.S. Pat. No. 4,942,126 discloses a method of identifying the presence of Campylobacter spp. in fecal specimens.
U.S. Pat. No. 5,200,344 discloses a method of identifying the presence of antibodies to C. jejuni or C. coli in test samples.
U.S. Pat. No. 5,374,531 discloses a method of analyzing particulate analytes, such as whole cells, in immunoassays.
U.S. Pat. No. 5,470,958 discloses an antigenic composition and antisera raised against a purified PEB 1 antigen from C. jejuni. 
U.S. Pat. Nos. 5,491,068 and 5,695,946 disclose a method of capturing specific bacterial cells with specific antibodies immobilized on magnetic beads.
U.S. Pat. No. 5,665,582 discloses a method for reversibly anchoring a biological material, such as a plastid, chromosome, nucleic acid, or protein to a solid support for use in an immunoassay.
U.S. Pat. No. 5,821,066 discloses a method of detecting, identifying, and quantifying respiring microorganisms in an immunoassay.
The present invention provides for an immunogen comprising outer membrane complexes from multiple strains of C. jejuni and C. coli, the immunogen inducing an immune response in animals, such as mice, enhancing the production of hybridomas specific for C. jejuni and C. coli epitopes, wherein the immunogen induces a broad range of antibodies against many outer membrane molecules of C. jejuni and C. coli, but not against other gram-negative enteric bacteria or non-jejuni/coli Campylobacter. Monoclonal antibodies of the present invention made by using such an immunogen are specific for epitopes expressed on C. jejuni and C. coli, wherein the monoclonal antibody binds the C. jejuni and C. coli porin protein or carbohydrate bound to porin protein, the porin comprising the 45 kD and 35 kD monomeric forms and the trimeric form of the porin. The invention also provides for monoclonal antibodies which bind specifically to a 43 kD protein expressed only on C. jejuni strains, or to carbohydrate bound to a 43 kD protein expressed only on C. jejuni strains.
The invention provides for a method of testing a sample for the presence of C. jejuni and/or C. coli comprising the steps of:
(a) exposing a sample suspected of containing C. jejuni and/or C. coli to a MAb which specifically binds C. jejuni and C. coli; 
(b) detecting MAb-antigen binding, said binding being indicative of the presence of C. jejuni and/or C. coli in said sample; and
(c) capturing C. jejuni or C. coli with MAb-conjugated-magnetic beads or MAb-conjugated-polystyrene beads or MAb conjugated or bound to a solid matrix, and detection of bound C. jejuni or C. coli with a method selected from: a second specific MAb, a PCR-specific assay, mass spectrometry (MALDI-TOF), reporter phage specific for C. jejuni or C. coli, optical sensing, or electronic sensing.
The invention also provides for a method of immunizing animals by:
(a) preparing an immunogenic complex by isolating outer membrane complexes from at least two Campylobacter strains selected from at least one C. jejuni and at least one C. coli; 
(b) pooling in approximately equivalent concentrations of the Campylobacter outer membrane complexes;
(c) immunizing animals intravenously; and
(d) screening antisera for Campylobacter antibody production.
The invention provides further for a method of making a monoclonal antibody specific for epitopes expressed on the outer membranes of C. jejuni and C. coli, wherein the monoclonal antibody binds a C. jejuni and C. coli porin protein or a porin protein-carbohydrate complex, the method comprising:
(a) preparing an immunogenic complex by isolating outer membrane complexes from at least two Campylobacter strains selected from the group consisting of C. jejuni and C. coli; 
(b) pooling in approximately equivalent concentrations of the Campylobacter outer membrane complexes;
(c) immunizing animals intravenously;
(d) screening antisera for Campylobacter antibody production;
(e) fusing isolated spleen cells from animals positive for Campylobacter antibody production and myeloma cells;
(f) isolating hybridomas making antibodies that bind Campylobacter; and
(g) screening and identifying monoclonal antibodies that bind Campylobacter.
The present invention further provides for monoclonal antibodies made by such a method, wherein the MAb binds common epitopes of C. jejuni and C. coli. 
The present invention is summarized further in that monoclonal antibodies specific for (1) unique epitopes of C. jejuni and (2) epitopes conserved between C. jejuni and C. coli outer membranes are produced by hybridomas formed by the fusion of cells from a mouse myeloma line and spleen cells from a mouse previously immunized with a mixture of outer membrane complexes (OMC) isolated from eight LPS serotype strains and two poultry strains of C. jejuni and C. coli. The monoclonal antibody designated as C731 is characterized in that it reacts with a protein having a molecular weight of approximately 44 kilodaltons and a carbohydrate moiety. The monoclonal antibody designated as C740 is characterized in that it reacts with a protein having a molecular weight of approximately 43 to 44 kilodaltons and a carbohydrate moiety.
The present invention is further directed to a process for producing monoclonal antibodies against Campylobacters comprising propagating a hybridoma formed by fusing a cell capable of producing antibodies against (1) C. jejuni or (2) C. jejuni and C. coli with a myeloma cell and harvesting the antibodies produced by the hybridoma.
It is an object of the present invention to produce monoclonal antibodies that are highly specific for (1) specific or distinct epitopes of C. jejuni and (2) epitopes shared between C. jejuni and C. coli outer membranes.
Another object of the present invention is to develop tests using monoclonal antibodies to assay for C. jejuni and C. coli isolated in pure culture and in clinical, food, and water samples.
It is further an object of the present invention to develop immunoassays for the detection of Campylobacter in food and in clinical specimens.
Another object of the present invention is to develop a procedure to isolate C. jejuni and C. coli from foods or other samples and to determine the prevalence of the organisms in the samples.
It is another object of the present invention to detect a neo-epitope of the outer membrane of C. jejuni, the neo-epitope defined here as an antigenic epitope comprising a porin protein-carbohydrate complex, wherein the neo-epitope is specific for C. jejuni and does not cross-react with other Campylobacters.
Because of their high specificity, the monoclonal antibodies may be a useful reagent for the detection of C. jejuni and/or C. coli in foods, clinical specimens, or in situ localization of the bacteria. The antibodies of the present invention may be used further to monitor environmental and/or waste water or various facilities for C. jejuni and/or C. coli contamination. The testing procedure would include, for example, enzyme-linked immunoassays (ELISAs), immunomagnetic capture, radioimmune assays, biosensor assays and other immunoassays including, but not limited to microscopic methods.
The monoclonal antibodies of the present invention may be used singly or in combination, such as in a cocktail mixture, to detect specific Campylobacter species, as neutralizing antibodies, or in a composition comprising one or more antibodies to be used in administering passive immunity to humans, livestock, poultry, or other animals.
The antibodies may be used further in side-by-side assays to determine whether the samples react only with the antibodies specific for both C. jejuni and C. coli or whether they react only with the antibodies that are specific for C. jejuni alone.
Another object of the invention is to provide an immunogen comprising outer membrane complexes from multiple strains of C. jejuni used as a vaccine which induces high levels of specific antibodies directed against C. jejuni and which protects against C. jejuni infection in humans, livestock, poultry, or other animals.
An additional object of the invention is to provide an immunogen comprising outer membrane complexes from multiple strains of C. jejuni and C. coli used as a vaccine which induces high levels of specific antibodies directed against both C. jejuni and C. coli and which protects against C. jejuni and C. coli infection in humans, livestock, poultry, or other animals.
Other objects and advantages of the invention will be apparent from the following detailed description and figures setting forth the preferred embodiment of the invention.